NF-κB2 Is a Putative Target Gene of Activated Notch-1 via RBP-Jκ

ABSTRACT

NF-κB2 (p100/p52), a member of the NF-κB/Rel family of transcription factors, is involved in the regulation of a variety of genes important for immune function. Previously, we have shown that the NF-κB2 gene is regulated in a positive and a negative manner. Two κB elements within the NF-κB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, we have shown that there exists a transcriptional repression in the absence of NF-κB. To identify a DNA binding activity responsible for this transcriptional repression, we have partially purified a nuclear complex, named Rep-κB. Here we further analyze this putative repressive binding activity. Detailed examination of Rep-κB–DNA interaction revealed the sequence requirements for binding to be almost identical to those of recombination signal binding protein Jκ (RBP-Jκ), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In addition, in electromobility shift assays, Rep-κB binding activity is recognized by an antibody directed against RBP-Jκ. By performing transient-transfection assays, we show that human RBP-Jκ represses basal as well as RelA (p65)-stimulated NF-κB2 promoter activity. Studies in Drosophila melanogaster have shown that Su(H) is implicated in the Notch signaling pathway regulating cell fate decisions. In transient-transfection assays we show that truncated Notch-1 strongly induces NF-κB2 promoter activity. In summary, our data clearly demonstrate that Rep-κB is closely related or identical to RBP-Jκ. RBP-Jκ is a strong transcriptional repressor of NF-κB2. Moreover, this repression can be overcome by activated Notch-1, suggesting that NF-κB2 is a novel putative Notch target gene.

ACKNOWLEDGMENTS

F.O. and S.L. contributed equally to this work.

We thank U. Wegenka for critically reading the manuscript. We also thank T. Henkel for providing the plasmid pSG5mNotch1IC, B. Ehring for the gift of pGST-TKmNotch1IC, and T. Honjo for providing the RBP-Jκ-specific antibody K0043 and the expression vector for murine RBP-2. For excellent technical assistance we thank E. Rüber and S. Schirmer. We also thank E. Ebert for providing reagents.

This study was supported by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 322 to S.L. and R.M.S. and by the Bundes- ministerium für Bildung und Forschung to R.M.S.